How to select the appropriate detection channel through the spectrogram?

There are a wide variety of fluorescent dyes used for Flow Cytometry. How do you know whether these fluorochrome are suitable for your Flow Cytometry, and which detection channel should be selected for each fluorochrome? This requires us to have a clear understanding of the fluorochrome used and the Flow Cytometry used for detection. We need to pay attention to the excitation wavelength and emission wavelength of fluorochrome, the laser and filter parameters of Flow Cytometry. The excitation wavelength and laser determine whether the target fluorochrome can be excited on the flow cytometer with specific configuration, and the emission wavelength and filter parameters determine whether the fluorescence of fluorochrome can be detected on the flow cytometer. Generally, fluorochrome with the maximum excitation wavelength near the laser wavelength can be excited.  When the maximum emission wavelength of fluorochrome falls within the detection range of the filter, its fluorescence can be detected by Flow Cytometry.

1. How to understand the excitation wavelength and emission wavelength of fluorochrome

We can better understand the wavelength information of fluorochrome through the spectrogram. Let's take FITC as an example to explain it in detail.

FITC Excitation and Emission Spectra

Excitation spectrum (Excitation, Ex): It refers to the light within a certain wavelength range that can specifically excite certain fluorochrome. Its peak is called excitation wave peak (maximum excitation wavelength, Ex-Max).

Emission spectrum (Emission, Em): It refers to the fluorescence within a certain wavelength range of fluorochrome emission caused by a certain wavelength excitation. Its peak is called emission wave peak (maximum emission wavelength, Em-Max).

Generally speaking, the energy of fluorescence emission light is lower than that of excitation light, and the wavelength of emission light is longer than that of excitation light. Fluorescent dyes can be excited to produce the strongest fluorescence at the maximum excitation wavelength in the excitation band. After obtaining the excitation wavelength of fluorochrome, the corresponding excitation light source can be selected for excitation. Knowing the emission wavelength of fluorochrome, you can select the corresponding filter at the maximum emission wavelength for detection.

2. Laser and filter

Take Beckman's CytoFLEX as an example, its instrument configuration is usually described as follows:

In the table, the excitation light wavelength "488 nm" means that the instrument is equipped with a 488 nm laser, and the fluorescence channel "525/40 BP" means that there is a band pass filter (BP, Band Pass) with a width of 40nm and a center of 525 nm under the 488nm laser, that is, a band pass filter with a detection wavelength range of 525 ± 20 nm.

3. Match fluorochrome with instrument

In the figure, the blue dotted line is the FITC excitation spectrum, its maximum excitation wavelength is 490 nm, and the red solid line is the FITC emission spectrum, its maximum emission wavelength is 530 nm. Therefore, FITC selects 488nm laser for excitation and 525/40 filter for detection.

The above is single laser detection. If the customer uses multi-color in the experiment, it is necessary to consider the interference of other fluorescence on this channel.

Note: The filters of different instruments may vary slightly, and the actual configuration of the instrument shall prevail.

Take Elab Fluor^® Violet 450 as an example: The optimal excitation wavelength of its fluorescence is 405 nm (the optimal excitation wavelength depends on the excitation of the flow cytometer laser), and the optimal detection channel is 450/45 (that is, the detection wavelength range is 450 ± 22.5 nm) band pass filter. It should be noted that if the emission wavelengths of fluorescent dyes are the same or similar, they are the same detection channel on the Flow Cytometry, and these fluorescent dyes cannot be used at the same time in multi-color experiments.